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Conservation Translocation (CT), which includes reintroduction, reinforcement and introduction, is gaining momentum, responding to serious population decline in many orchids. Orchid conservation underpinned by a greater emphasis on understanding biotic and abiotic factors of habitats is critical for successful recovery and restoration programmes. Anacamptis morio, commonly known as green-winged orchid (GWO), is a terrestrial orchid found throughout Europe, but populations in England rapidly declined in its native range in the last several decades. The current study explored the relationship between soil abiotic characteristics and the community composition of key fungal groups. Wild sites in Essex and Cumbria in England, where successful colonies are currently present, were compared to potential sites for CT in Cumbria. The Cumbrian sites managed by cattle grazing include the wild site and three potential CT sites, with two of them hosting no GWO plants. The Essex site, fOxley Meadow, where no cattle is used for grassland management, hosts the largest population of GWO in England. The aim of this study was to understand whether the community composition of fungi and soil characteristics of the potential CT sites in Cumbria are nearly compatible with that of Oxley Meadow. Oxley Meadow, with around 65,000 plants, stands out as a unique habitat compared to all Cumbria sites, as it showed low organic content. Nitrate and phosphate content were smaller for Oxley Meadow compared to other Cumbrian sites. The proportion of Basidiomycota fungi was greater in Oxley Meadow compared to all Cumbrian sites where Ascomycota dominated. The abundant fungal group found in Oxley Meadow was Agaricales. From Agariclaes, Hygrophoraceae or waxcaps fungi are considered an indicator group of fungi and were the most abundant group in Oxley Meadow. They have a negative correlation with targeted key fungal groups and abiotic parameters. More in-depth assessments using additional primers are essential to better understand the fungal diversity and how this diversity translates to the resilience of orchid habitats. This preliminary study points to future studies to assess whether sites are near-compatible to the wild site where large colonies are present using additional primers collected over different time scales. [ABSTRACT FROM AUTHOR]

Entomopathogenic fungi are important component for regulation of pests in ecosystem. Isaria fumosorosea, as one of the entomopathogenic fungi, was reported to successfully controlled the outbreaks of forest defoliators attacked larch (Larix kaempferi) plantation in Furano, Japan and beech (Fagus crenata) forest in Hachimantai, Japan. Instead of semi-cultured method, in this paper, a culture-independent method based on DNA using qPCR was developed for specific detection and quantification of I. fumosorosea directly from soil DNA extract using specific primer. The primer IFU5821F/IFU6061R was designed and found to be the best primer pair for I. fumosorosea. Standard soil DNA was obtained with strong relationship and good fitting with five levels (R2= 0.989, E = 0.58). I. fumosorosea could not be detected from all soil samples which was possibility caused by low density of the fungi. The qPCR was likely a rapid and specific method to detect the fungus from soil.

Conservation Translocation (CT), which includes reintroduction, reinforcement and introduction, is gaining momentum, responding to serious population decline in many orchids. Orchid conservation underpinned by a greater emphasis on understanding biotic and abiotic factors of habitats is critical for successful recovery and restoration programmes. Anacamptis morio, commonly known as green-winged orchid (GWO), is a terrestrial orchid found throughout Europe, but populations in England rapidly declined in its native range in the last several decades. The current study explored the relationship between soil abiotic characteristics and the community composition of key fungal groups. Wild sites in Essex and Cumbria in England, where successful colonies are currently present, were compared to potential sites for CT in Cumbria. The Cumbrian sites managed by cattle grazing include the wild site and three potential CT sites, with two of them hosting no GWO plants. The Essex site, fOxley Meadow, where no cattle is used for grassland management, hosts the largest population of GWO in England. The aim of this study was to understand whether the community composition of fungi and soil characteristics of the potential CT sites in Cumbria are nearly compatible with that of Oxley Meadow. Oxley Meadow, with around 65,000 plants, stands out as a unique habitat compared to all Cumbria sites, as it showed low organic content. Nitrate and phosphate content were smaller for Oxley Meadow compared to other Cumbrian sites. The proportion of Basidiomycota fungi was greater in Oxley Meadow compared to all Cumbrian sites where Ascomycota dominated. The abundant fungal group found in Oxley Meadow was Agaricales. From Agariclaes, Hygrophoraceae or waxcaps fungi are considered an indicator group of fungi and were the most abundant group in Oxley Meadow. They have a negative correlation with targeted key fungal groups and abiotic parameters. More in-depth assessments using additional primers are essential to better understand the fungal diversity and how this diversity translates to the resilience of orchid habitats. This preliminary study points to future studies to assess whether sites are near-compatible to the wild site where large colonies are present using additional primers collected over different time scales.

The relationship between the concentration of soil DNA and microbial biomass determined by the method of substrate-induced respiration was studied in a wide range of soils differing in particle size distribution, acidity, organic carbon content, microbial biomass, and type of human activity in antiquity and at the present time. Calcaric Leptosols and Leptic Phaeozems of medieval agricultural lands in the Central Caucasus, Stratozems of agricultural terraces of the late Middle Ages—Modern times in the middle mountain zone of the Eastern Caucasus, as well as Kastanozems and Solonetzes with different grazing intensity in the dry steppe zone (Rostov region) were chosen as the key sites. It was shown that the determination of soil double-stranded DNA concentration is a reliable and simple method for determining microbial biomass in soils with a loamy texture, the organic carbon content < 2%, and the microbial biomass <700 µg C/g dry soil. The conversion factor FDNA in such soils varied in a narrow range from 5.24 to 5.41. In soils with a high content of organic carbon, an increase in FDNA (6.56 and 10.56) was observed due to the presence of recalcitrant extracellular DNA. Agristratified soil of sandy loamy texture was characterized by a lower degree of double-stranded DNA preservation, which resulted in a decrease in the determined microbial biomass (FDNA = 4.22). A reduced conversion factor FDNA (4.78) was also found in the soils of pastures in the dry steppe zone, which confirms the known limitations of using the substrate-induced respiration method in alkaline soils. Human activity did not significantly affect the relationship between the amount of soil DNA and microbial biomass. [ABSTRACT FROM AUTHOR]

Based on the new generation sequencing methods, such as pyrosequencing (Roche/454 Life Sciences), the metagenomic characteristics of typical chernozem microbiomes were determined and described for the first time in Moldova under different land use conditions. It was revealed that the community of prokaryotes is represented by both domains, particularly twelve bacterial phyla (61.4%) and one phylum Archaea (6.2%). Only nine phyla had an abundance above 1.0%: the archaeal phylum Сrenarchaeota (Thaumarchaeota) and the bacterial phyla Proteobacteria, Actinobacteriota, Bacteroidota, Acidobacteriota, Firmicutes, Verrumicrobiota, Platctomycecota, and Myxococcota. Phyla, with a share of less than 1.0%, preferred the conditions of experimental variants of crop rotations. A significant proportion were unidentified microorganisms, the abundance of which varied from 25.8 to 38.2% in spring and from 27.9 to 40.2% in summer. The influence of soil and climatic conditions on the formation of the typical chernozem microbiome was more significant than agricultural practices.

Entomopathogenic fungus are important component in pests regulation in ecosystem. Isaria fumosorosea, as one of the entomopathogenic fungi, was reported to successfully control outbreaks of forest defoliators known to attack larch (Larix kaempferi) plantation in Furano, Japan and beech (Fagus crenata) forest in Hachimantai, Japan. Instead of semi-cultured methods, this paper developed and used a culture-independent method based on DNA using qPCR for specific I. fumosorosea detection and quantification directly from soil DNA extraction using specific primer. The primer IFU5821F/IFU6061R was designed and the best primer pair for I. fumosorosea. Strong relationship and good fitting with five levels (R2 = 0.989, E = 0.58) were obtained standard soil DNA extraction. I. fumosorosea could not be detected from all soil samples possibly caused by low density of the fungi. The qPCR was likely a rapid and specific method to detect the fungus from soil. [ABSTRACT FROM AUTHOR]

Abstract Background Advances in DNA sequencing technologies have transformed our capacity to perform life science research, decipher the dynamics of complex soil microbial communities and exploit them for plant disease management. However, soil is a complex conglomerate, which makes functional metagenomics studies very challenging. Results Metagenomes were assembled by long-read (PacBio, PB), short-read (Illumina, IL), and mixture of PB and IL (PI) sequencing of soil DNA samples were compared. Ortholog analyses and functional annotation revealed that the PI approach significantly increased the contig length of the metagenomic sequences compared to IL and enlarged the gene pool compared to PB. The PI approach also offered comparable or higher species abundance than either PB or IL alone, and showed significant advantages for studying natural product biosynthetic genes in the soil microbiomes. Conclusion Our results provide an effective strategy for combining long and short-read DNA sequencing data to explore and distill the maximum information out of soil metagenomics.

The analysis of 18S rRNA gene amplicons is an important tool to characterize the diversity of the eukaryotic soil microbiome. Here we analyzed two primer sets (TAReuk, EKeuk) and the impact of a newly designed antifungal peptide nucleic acid to enhance the detection of protists in silico and with soil DNA extracted from croplands and a forest. The in silico analyses showed for TAReuk pronounced specificities for protist SAR supergroup and Metazoa, while EKeuk was particularly specific for Ascomycota and Basidiomycota. In silico , the PNA matched with the majority of Ascomycota (81.3 %) and Basidiomycota (65.4 %), but with <6 % of protists. The contrasting primer specificities were confirmed with soil DNA, but the proportion of protist amplicons was similar. In contrast to in silico , effects of the PNA were not as clear with soil DNA, even though it completely inhibited the amplification of the targeted fungal sequences. PNA effects were more pronounced with TAReuk, and results with cropland and forest soil DNA were not consistent, e.g. , for cropland, PNA decreased the relative abundance of fungi but for forest it was the opposite, possibly because of different fungal diversity. The divergence between PNA in silico -predictions and results with soil DNA are likely an outcome of primer binding to <100 % complementary target sequences and a still limited DNA sequence databases for soil microbial eukaryotes. With TAReuk, the presence of PNA enhanced the detection of Conosa and, thus, could be a useful tool to study this group in the future. • A new PNA probe inhibited in silico PCR amplification of most fungal 18S rRNA genes. • With soil DNA, the PNA probe did not inhibit amplification of fungal 18S rRNA genes. • PNA effects on detection of protists varied between two 18S rRNA primer pairs. • PNA effects varied between DNA extracted from five different soils. • With the common TAReuk, protist primers PNA increased the detection of Conosa. [ABSTRACT FROM AUTHOR]

Genetic fingerprinting demonstrated in previous studies that differently sized soil particle fractions (PSFs; clay, silt, and sand with particulate organic matter (POM)) harbor microbial communities that differ in structure, functional potentials and sensitivity to environmental conditions. To elucidate whether specific bacterial or archaeal taxa exhibit preference for specific PSFs, we examined the diversity of PCR-amplified 16S rRNA genes by high-throughput sequencing using total DNA extracted from three long-term fertilization variants (unfertilized, fertilized with minerals, and fertilized with animal manure) of an agricultural loamy sand soil and their PSFs. The PSFs were obtained by gentle ultrasonic dispersion, wet sieving, and centrifugation. The abundance of bacterial taxa assigned to operational taxonomic units (OTUs) differed less than 2.7% between unfractionated soil and soil based on combined PSFs. Across the three soil variants, no archaeal OTUs, but many bacterial OTUs, the latter representing 34–56% of all amplicon sequences, showed significant preferences for specific PSFs. The sand-sized fraction with POM was the preferred site for members of Bacteroidetes and Alphaproteobacteria, while Gemmatimonadales preferred coarse silt, Actinobacteria and Nitrosospira fine silt, and Planctomycetales clay. Firmicutes were depleted in the sand-sized fraction. In contrast, archaea, which represented 0.8% of all 16S rRNA gene sequences, showed only little preference for specific PSFs. We conclude that differently sized soil particles represent distinct microenvironments that support specific bacterial taxa and that these preferences could strongly contribute to the spatial heterogeneity and bacterial diversity found in soils.

Genetic fingerprinting demonstrated in previous studies that differently sized soil particle fractions (PSFs; clay, silt, and sand with particulate organic matter (POM)) harbor microbial communities that differ in structure, functional potentials and sensitivity to environmental conditions. To elucidate whether specific bacterial or archaeal taxa exhibit preference for specific PSFs, we examined the diversity of PCR-amplified 16S rRNA genes by high-throughput sequencing using total DNA extracted from three long-term fertilization variants (unfertilized, fertilized with minerals, and fertilized with animal manure) of an agricultural loamy sand soil and their PSFs. The PSFs were obtained by gentle ultrasonic dispersion, wet sieving, and centrifugation. The abundance of bacterial taxa assigned to operational taxonomic units (OTUs) differed less than 2.7% between unfractionated soil and soil based on combined PSFs. Across the three soil variants, no archaeal OTUs, but many bacterial OTUs, the latter representing 34-56% of all amplicon sequences, showed significant preferences for specific PSFs. The sand-sized fraction with POM was the preferred site for members of Bacteroidetes and Alphaproteobacteria, while Gemmatimonadales preferred coarse silt, Actinobacteria and Nitrosospira fine silt, and Planctomycetales clay. Firmicutes were depleted in the sand-sized fraction. In contrast, archaea, which represented 0.8% of all 16S rRNA gene sequences, showed only little preference for specific PSFs. We conclude that differently sized soil particles represent distinct microenvironments that support specific bacterial taxa and that these preferences could strongly contribute to the spatial heterogeneity and bacterial diversity found in soils. [ABSTRACT FROM AUTHOR]

The effects of different tillage systems on the properties and microbial diversity of an agricultural soil was investigated. In doing so, soil physical, chemical and biological properties were analysed in 2013-2015, on a long-term field experiment on a loamy sand at the IUNG-PIB Experimental Station in Grabów, Poland. Winter wheat was grown under two tillage treatments: conventional tillage using a mouldboard plough and traditional soil tillage equipment, and reduced tillage based on soil crushing-loosening equipment and a rigid-tine cultivator. Chopped wheat straw was used as a mulch on both treatments. Reduced tillage resulted in increased water content throughout the whole soil profile, in comparison with conventional tillage. Under reduced tillage, the content of readily dispersible clay was also reduced, and, therefore, soil stability was increased in the toplayers, compared with conventional tillage. In addition, the beneficial effects of reduced tillage were reflected in higher soil microbial activity as measured with dehydrogenases and hydrolysis of fluorescein diacetate, compared with conventional tillage. Moreover, the polimerase chain reaction - denaturing gradient gel electrophoresis analysis showed that soil under reduced tillage had greater diversity of microbial communities, compared with conventionally-tilled soil. Finally, reduced tillage increased organic matter content, stability in water and microbial diversity in the top layer of the soil. [ABSTRACT FROM AUTHOR]

Plant soil feedbacks ( PSFs) are thought to be important to plant growth and species coexistence, but most support for these hypotheses is derived from short-term greenhouse experiments. Here we use a seven-year, common garden experiment to measure PSFs for seven native and six nonnative species common to the western United States. We use these long-term, field-based estimates to test correlations between PSF and plant landscape abundance, species origin, functional type, and lifespan. To assess potential PSF mechanisms, we also measured soil microbial community composition, root biomass, nitrogen cycling, bulk density, penetration resistance, and shear strength. Plant abundance on the landscape and plant lifespan were positively correlated with PSFs, though this effect was due to the relationships for native plants. PSFs were correlated with indices of soil microbial community composition. Soil nutrient and physical traits and root biomass differed among species but were not correlated with PSF. While results must be taken with caution because only 13 species were examined, these species represent most of the dominant plant species in the system. Results suggest that native plant abundance is associated with the ability of long-lived plants to create positive plant-soil microbe interactions, while short-lived nonnative plants maintain dominance by avoiding soil-borne antagonists, increasing nitrogen cycling and dedicating resources to aboveground growth and reproduction rather than to belowground growth. Broadly, results suggest that PSFs are correlated with a suite of traits that determine plant abundance. [ABSTRACT FROM AUTHOR]

Soil complexity, heterogeneity and transferability make it valuable in forensic investigations to help obtain clues as to the origin of an unknown sample, or to compare samples from a suspect or object with samples collected at a crime scene. In a few countries, soil analysis is used in matters from site verification to estimates of time after death. However, up to date the application or use of soil information in criminal investigations has been limited. In particular, comparing bacterial communities in soil samples could be a useful tool for forensic science. To evaluate the relevance of this approach, a blind test was performed to determine the origin of two questioned samples (one from the mock crime scene and the other from a 50:50 mixture of the crime scene and the alibi site) compared to three control samples (soil samples from the crime scene, from a context site 25m away from the crime scene and from the alibi site which was the suspect's home). Two biological methods were used, Ribosomal Intergenic Spacer Analysis (RISA), and 16S rRNA gene sequencing with Illumina Miseq, to evaluate the discriminating power of soil bacterial communities. Both techniques discriminated well between soils from a single source, but a combination of both techniques was necessary to show that the origin was a mixture of soils. This study illustrates the potential of applying microbial ecology methodologies in soil as an evaluative forensic tool. [ABSTRACT FROM AUTHOR]

The polymorphisms of soil fungal rDNA Internal Transcribed Spacer regions were studied in Korean pine forests of various ages (10-100-year-old trees) by means of cloned libraries, and analyzed to determine the effects of the trees' developmental stage on soil fungal community structure. The obtained Shannon diversity index (H) and richness (S) indicated that the diversity of the soil fungal community increased significantly with the development of Korean pines ( P < 0.05). In addition, cluster analysis (UPGMA) showed that the soil fungal community variety associated with differently aged Korean pines was higher than 50 %. The soil fungal community diversity correlated significantly with the N content and C/N ratio of the soil ( P < 0.05). The results of this study indicate that the age of in Korean pine can affect soil fungal community by altering soil properties, which in turn could affect the nutrient cycling in the forest ecosystem. [ABSTRACT FROM AUTHOR]

For the first time, the composition of microbiomes in the biological crust (AKL) horizons of extremely arid desert soils (Aridic Calcisols) developed from saline and nonsaline alluvial deposits in the Ili Depression (eastern Kazakhstan) was analyzed. To describe the diversity of microorganisms in the soil samples, a novel method of pyrosequencing (Roche/454 Life Sciences) was applied. It was shown that bacteria from the Proteobacteria, Actinobacteria, Firmicutes, Verrucomicrobia, Acidobacteria, and Bacteroidetes phyla predominate in all the samples; these are typical representatives of the microbiome of soil crusts. A distinctive feature of the extremely arid soils is the high contribution of cyanobacteria (25-30%) to the total DNA. In the soils developed from saline sediments, representatives from the Rubrobacteraceae, Streptococcaceae, and Caulobacteraceae families and from the Firmicutes phylum predominated. In the soils developed from nonsaline gypsiferous deposits, bacteria from the class of Acidobacteria, subgroup Gp3, of the Methylobacteriaceae family and the class of Subdivision 3 from the Verrucomicrobia phylum predominated. [ABSTRACT FROM AUTHOR]

A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils. [ABSTRACT FROM AUTHOR]

The objective of this study was to characterize the microbial communities attached to clay (< 2 μm), fine silt (2-20 μm), coarse silt (20-63 μm) and sand-sized fractions [> 63 μm; including particulate organic matter ( POM)] of an arable soil and analyse their response to more than 100 years of two different fertilization regimes. Mild ultrasonic dispersal, wet-sieving and centrifugation allowed the separation of soil particles with the majority of bacterial cells and DNA still attached. Fertilizations increased soil organic carbon ( SOC), total DNA and the abundance of bacterial, archaeal and fungal r RNA genes more strongly in the larger-sized fractions than in fine silt, and no effect was seen with clay, the latter representing above 70% of the total microbial populations. A highly positive correlation was found between microbial r RNA genes and the surface area provided by the particles, while the correlation with SOC was lower, indicating a particle-size-specific heterogeneous effect of SOC. The prokaryotic diversity responded more strongly to fertilization in the larger particles but not with clay. Overall, these results demonstrate that microbial responsiveness to long-term fertilization declined with smaller particle sizes and that especially clay fractions exhibit a high buffering capacity protecting microbial cells against changes even after 100 years under different agricultural management. [ABSTRACT FROM AUTHOR]

The conservation of rare fungal sites occurring on actively managed forest lands requires efficient site monitoring methods. In this study species-specific primers for the putatively mycorrhizal Albatrellus ellisii (Russulales) were developed so that DNA extracted from soil samples at known sites of this fungus could be tested for the presence of A. ellisii mycelium with PCR. This method was used to measure seasonal changes in the levels of A. ellisii mycelium at three study sites while the utility of this monitoring method was assessed. We found that A. ellisii maintained a constant level of soil occupancy over three seasons, except at one site where mycelium abundance increased in the fall. No reduction in abundance was seen in the summer, although all three sites experienced significant summer drought. We found species-specific genetic marker detection to be an effective and practical method for monitoring the mycelial distribution of a target fungus in soil. The ability to obtain this data from rare fungal sites advances our capability to conserve these fungi, particularly within the managed forest landscape. [ABSTRACT FROM AUTHOR]

Abstract: Most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches promise the accessibility of the genetic resources and their potential applications. Genetic resources from terrestrial environments can be accessed by exploring the soil metagenome. Soil metagenomic analyses are usually initiated by the isolation of environmental DNAs. Several methods have been described for the direct isolation of environmental DNAs from soil and sediments. Application of metagenomics largely depends on the construction of genomic DNA libraries and subsequent high-throughput sequencing or library screening. Thus, obtaining large quantities of pure cloneable DNA from the environment is a prerequisite. This review discusses the recent developments related to efficient extraction and purification of soil metagenome highlighting the considerations for various metagenomic applications. [Copyright &y& Elsevier]

Abstract: Agroforestry and grass buffers have been shown to improve soil properties and overall environmental quality. The objective of this study was to examine management and landscape effects on water stable soil aggregates (WSA), soil carbon, soil nitrogen, enzyme activity, and microbial community DNA content. Treatments were row crop (RC), grass buffer (GB), agroforestry buffer (AG), and grass waterways (GWW). A corn (Zea mays L.)–soybean (Glycine max L.) rotation under no-till management was established in a watershed in northeast Missouri in 1991; grass buffers were implemented in 1997. Grass buffers, 4.5m wide and 36.5m apart, consisted of a mixture of redtop (Agrostis gigantea Roth), brome grass (Bromus spp.), and birdsfoot trefoil (Lotus corniculatus L.) on contour within the watershed. Agroforestry buffers have pin oak (Quercus palustris Muenchh.) trees distributed down the center of the grass buffers on one half of the watershed. Soils were collected from two transects extending from the summit to lower landscape positions within the grass and agroforestry portions of the watershed in June 2006. Soil enzymes studied include: fluorescein diacetate hydrolase, β-glucosidase, glucosaminidase, and dehydrogenase. Soil DNA content was determined as an alternative for microbial biomass. WSA was significantly different among treatments and landscape positions. WSA decreased from GWW>AG>GB>RC management treatments and also decreased from lower>middle>summit landscape positions. Soil carbon and nitrogen were highest for the GWW treatment and lowest for RC. The landscape position effect was significant for RC and AG treatments. Fluorescein diacetate, β-glucosidase and glucosaminidase enzyme activities were significantly higher in buffers and GWW areas than RC areas. Dehydrogenase activity was different between grass (GB and GWW) and crop areas. The landscape effect was insignificant for enzyme activity. Although soil DNA may be a good indicator of microbial biomass, it did not appear to differentiate among management systems as selectively as other microbial parameters. Results of the study show that establishment of AG, GB, and GWW increased WSA, soil carbon, soil nitrogen, and enzyme activity. [Copyright &y& Elsevier]

Abstract: The impacts of tillage regime, cover crop, and nitrogen on various soil organisms inhabiting a fragile sandy soil (Brunosolic Gray Brown Luvisol) were determined. Soil samples were collected between 2000 and 2003 from a long-term tillage experiment, established in 1988 to determine the effect of tillage systems on yield of corn (Zea mays), soil quality, and weed populations. Populations of several of the soil organisms studied were significantly affected by one or more agronomic treatments. A single earthworm species, Aporrectodea turgida, was found in the experimental area. Worm populations were generally low and dominated by juveniles. Spring-sampled populations were significantly higher in no-till plots than in conventionally tilled plots. Fall-sampled populations were not affected as greatly by tillage, but were generally higher in no-till plots not receiving additional nitrogen or in plots overseeded with a rye (Secale cereale) cover crop. Soil microbial biomass, as represented by extractable soil DNA, was higher in the spring than in the fall. Populations of the soilborne stramenopile Pythium were generally higher in conventionally tilled plots, and were increased by a rye cover crop. Higher rates of nitrogen increased populations of total soil fungi but nitrogen had little effect on prostigmatid or cryptostigmatid mites; prostigmatid populations were generally higher in no-till plots. Spring populations of mesostigmatid mites were higher in plots with a rye cover crop than in plots without an overwintering plant cover. Conventional tillage stimulated populations of astigmatid mites during periods of high rainfall. Collembola populations were dominated by the families Onychiuridae and Isotomidae, but neither was greatly affected by any tillage treatment. Principal component analysis showed that populations of A. turgida and soil aggregation tended to be positively associated with one another, but that variations in populations of Onychiuridae springtails, prostigmatid mites, and Pythium tended not to be associated with changes in other variables. Overall, effects of tillage treatments on soil organisms were found to differ from previous reports in several respects, suggesting that soil type may impose conditions that over-ride the impacts of agronomic cultivation systems on populations of soil organisms. [Copyright &y& Elsevier]

We tested whether fungal communities are impacted by nitrogen deposition or increased precipitation by PCR-amplifying partial fungal rRNA genes from 24 soil and 24 root samples from a nitrogen enrichment and irrigation experiment in a tallgrass prairie at Konza Prairie Biological Station in northeastern Kansas, U.S.A. Obtained fungal sequences represented great fungal diversity that was distributed mainly in ascomycetes and basiodiomycetes; only a few zygomycetes and glomeromycetes were detected. Conservative extrapolated estimates of the fungal species richness suggest that the true richness may be at least twice as high as observed. The effects of nitrogen enrichment or irrigation on fungal community composition, diversity or clone richness could not be unambiguously assessed because of the overwhelming diversity. However, soil communities differed from root communities in diversity, richness and composition. The compositional differences were largely attributable to an abundant, soil-inhabiting group placed as a well-supported sister group to other ascomycetes. This group likely represents a novel group of fungi. We conclude that the great fungal richness in this ecosystem precluded a reliable assessment of anthropogenic impacts on soil or rhizosphere communities using the applied sampling scheme, and that detection of novel fungi in soil may be more a rule than an exception. [ABSTRACT FROM AUTHOR]

Examines the cloning of aminoglycoside-biosynthetic genes from soil DNA for accumulation of genetic information. Identification of aminotransferase genes; Phylogenetic analysis of the genes; Occurrence of horizontal gene transfer in the soil; Biosynthesis of aminoglycoside antibiotics.

In most studies, the distribution of the mycelia of ectomycorrhizal fungi in forest soils has been inferred from areas occupied by fruit bodies. Here, we investigated the below-ground spatio-temporal distribution of Hebeloma cylindrosporum by polymerase chain reaction quantification of a specific DNA sequence present in DNA extracted from soil. Soil samples were collected in a Pinus pinaster stand located in a campsite where fruit bodies of H. cylindrosporum had been mapped from 1990 to 2000. In samples collected underneath fruit bodies, DNA of H. cylindrosporum was always detected in large amounts. However, this DNA was often undetectable in the absence of fruit body even 1 yr after their disappearance. This result was supported by the failure to identify mycorrhizas of this species on seedlings planted in soil samples collected in places where fruit bodies were present 1 yr before sampling. This pattern suggests a recolonization of the site each year by H. cylindrosporum basidiospores in ‘receptive’ patches of ground created either by the local elimination of competitors or by local nutrient enrichments, which could frequently occur in a campsite. Our results demonstrate that an ectomycorrhizal species can be completely eliminated from the roots within 1 yr and does not necessarily contribute to the next generation of mycorrhizas. [ABSTRACT FROM AUTHOR]

In natural forest ecosystems several ectomycorrhizal fungal species cohabit on host plant root systems. To evaluate the ecological and functional impact of each species, it is necessary to appreciate the distribution and abundance of its mycelia in the soil. We developed a competitive PCR (cPCR) method for the basidiomycete Hebeloma cylindrosporum that allows quantification of its DNA in complex DNA mixtures extracted directly from soil samples. The target sequence chosen for the cPCR analysis was a 533-bp fragment of the nuclear ribosomal intergenic spacer, amplified using two species-specific primers. The detection threshold of the cPCR protocol developed was 0.03 pg of genomic DNA. This method was applied to soil samples collected from beneath and at various distances from a group of fruit bodies in a Pinus pinaster forest stand. The results revealed that H. cylindrosporum below-ground biomass was concentrated directly underneath the fruit bodies or very close to them, while no DNA of this species could be detected in soil samples collected at more than 50 cm away. In the vicinity of fruit bodies, H. cylindrosporum soil DNA concentration varied considerably (between 10 and 0.07 ng g soil−1) and decreased sharply with increased distance from the fruit bodies. This work demonstrates the potential of competitive quantitative PCR for the study of the distribution, abundance and persistence of the mycelia of an ectomycorrhizal fungal species in soil. [Copyright &y& Elsevier]

Actinomycetes play a relevant role in soil ecology and are also of important biotechnological interest as they produce several bioactive metabolites. Within the filamentous actinomycetes, it would be desirable to recognize and characterize environmental samples containing unusual genera. To this end, we have developed selective primer sets for polymerase chain reaction (PCR) amplification of 16S rDNA from the Actinomycetales families Micromonosporaceae, Streptomycetaceae, Streptosporangiaceae and Thermomonosporaceae, and from the genus Dactylosporangium. Each primer set, evaluated on genomic DNA from reference strains, showed high specificity and good sensitivity. After amplification of DNA extracted from soil samples, the sequence of the cloned PCR products confirmed the specific amplification of the desired group of sequences in at least 95% of the clones for each primer set. The application of these primers to environmental samples showed the frequent occurrence of these groups in soil samples and also revealed sequences that can be attributed to new groups of actinomycetes. [Copyright &y& Elsevier]

Soil microbes exert critical control over ecosystem functions by regulating organic matter decomposition and nutrient cycling. Diverse methods have been adopted to estimate the soil microbial biomass carbon (C mic) and nitrogen (N mic), largely based on fumigation and respiration or biomarkers, such as phospholipids and ATP. Although soil microbial DNA is also an important biomarker, whether this biomarker can be used to estimate C mic and N mic at a large scale in arid and semi-arid regions that remains unclear. To explore this, the 124 soil samples collected from 28 different vegetation/microhabitat types in diverse ecosystems (i.e., desert, cropland, typical steppe, meadow steppe and forest) across arid and semi-arid regions in northern China; then, the strong linear relationships were developed among soil microbial DNA concentrations with the C mic and N mic contents estimated by the classic chloroform fumigation-extraction (CFE) method. The results showed that the soil microbial DNA concentrations were linearly related to the observed C mic (r = 0.99) and N mic (r = 0.97) across the diverse vegetation types, although soil microbial biomass presented an enormous fluctuation. Moreover, based on the above-mentioned coefficients of the linear relationship, it was further inferred that the mean proportions of carbon and nitrogen elements in DNA were 0.99% and 3.70% for C mic and N mic , respectively, when assuming the G-C base-pair proportion is 50% in double-stranded DNA. In conclusion, the study proved that soil microbial DNA as a powerful indicator may provide convenient and efficient estimates for C mic and N mic in arid and semi-arid systems. Although this method still needs to be further improved and examined at a larger scale, it provides a good addition to the already existing methods to determine microbial biomass. Unlabelled Image [ABSTRACT FROM AUTHOR]